Deciphering bat influenza H18N11 infection dynamics in male Jamaican fruit bats on a single-cell level.

Jamaican fruit bats (Artibeus jamaicensis) naturally harbor a wide range of viruses of human relevance. These infections are typically mild in bats, suggesting unique features of their immune system. To better understand the immune response to viral infections in bats, we infected male Jamaican fruit bats with the bat-derived influenza A virus (IAV) H18N11. Using comparative single-cell RNA sequencing, we generated single-cell atlases of the Jamaican fruit bat intestine and mesentery. Gene expression profiling showed that H18N11 infection resulted in a moderate induction of interferon-stimulated genes and transcriptional activation of immune cells. H18N11 infection was predominant in various leukocytes, including macrophages, B cells, and NK/T cells. Confirming these findings, human leukocytes, particularly macrophages, were also susceptible to H18N11, highlighting the zoonotic potential of this bat-derived IAV. Our study provides insight into a natural virus-host relationship and thus serves as a fundamental resource for future in-depth characterization of bat immunology.

3D approaches to culturing bovine skin: explant culture versus organotypic skin model.

INTRODUCTION: Digital Dermatitis (DD) in cattle appears with high prevalence, nevertheless the knowledge on its pathogenesis is still limited. In this context, in vitro skin models represent a valuable tool to facilitate the study of DD. METHODS: Two in vitro skin models were established using bovine distal limb skin: a skin explant model and an organotypic skin model. For the skin explant model, skin samples were cultured with an air-liquid interface for up to 7 days. Besides routine histopathological examination, read-out parameters were Ki-67 and cleaved Caspase-3 stainings. For the organotypic model, primary keratinocytes were layered on top of a dermal equivalent containing mainly mitotically inactive fibroblasts and maintained for up to 21 days. At regular intervals (day 7, 14 and 21), cultured skin samples were taken for (immuno)histological analysis. RESULTS: Both cultures could be maintained for the entire duration of the intended culture period. In the histopathological assessment, explant skin cultures showed ballooning degeneration of keratinocytes and segmental necrosis starting at day 5 of culturing. Initially basal keratinocytes in the organotypic model differentiated as demonstrated by positive Keratin 14, Desmoglein-1, Loricrin and Involucrin immunofluorescent stainings. Ki-67 was observed occasionally and suprabasally still after 21 days of culture. CONCLUSION: Both in vitro models proved dependable and constitute a viable option for replacing experiments on live animals, each with its own benefits. Whereas skin explants include all cell types available in vivo and can therefore reflect realistic cell-cell-interactions and signaling pathways, the organotypic model offers a higher standardization and reproducibility. Depending on the focus of future studies, both models can be used for specific experimental purposes of bovine dermatological research in general or specialized questions concerning (infectious) claw diseases as e.g. DD.

Phosphatidylinositolmannoside vaccination induces lipid-specific Th1-responses and partially protects guinea pigs from Mycobacterium tuberculosis challenge.

The concept of donor-unrestricted T cells (DURTs) comprises a heterogeneity of lymphoid cells that respond to an abundance of unconventional epitopes in a non-MHC-restricted manner. Vaccinologists strive to harness this so far underexplored branch of the immune system for new vaccines against tuberculosis. A particular division of DURTs are T cells that recognize their cognate lipid antigen in the context of CD1-molecules. Mycobacteria are characterized by a particular lipid-rich cell wall. Several of these lipids have been shown to be presented to T cells via CD1b-molecules. Guinea pigs functionally express CD1b and are hence an appropriate small animal model to study the role of CD1b-restricted, lipid-specific immune responses. In the current study, guinea pigs were vaccinated with BCG or highly-purified, liposome-formulated phosphatidylinositol-hexa-mannoside (PIM6) to assess the effect of CD1-restricted DURTs on the course of infection after virulent Mycobacterium tuberculosis (Mtb) challenge. Robust PIM6-specific T cell-responses were observed both after BCG- and PIM6-vaccination. The cellular response was significantly reduced in the presence of monoclonal, CD1b-blocking antibodies, indicating that a predominant part of this reactivity was CD1b-restricted. When animals were challenged with Mtb, BCG- and PIM6-vaccinated animals showed significantly reduced pathology, smaller necrotic granulomas in lymph node and spleen and reduced bacterial loads. While BCG conferred an almost sterile protection in this setting, compared to control animals' lesions were reduced roughly by two thirds in PIM6-vaccinated. Comprehensive histological and transcriptional analyses in the draining lymph node revealed that protected animals showed reduced transcription-levels of inflammatory cyto- and chemokines and higher levels of CD1b-expression on professional antigen cells compared to controls. Although BCG as a comparator induced by far stronger effects, our observations in the guinea pig model suggest that CD1b-restricted, PIM6-reactive DURTs contribute to immune-mediated containment of virulent Mtb.

Pyelonephritic end-stage kidney and ureterocutaneous fistula in a Shetland pony.

A 12-y-old Shetland Pony was presented with a mucus-secreting fistula in the right paralumbar fossa. Surgery was performed to unravel the origin of the fistula. The horse died under anesthesia and was forwarded to autopsy. The right kidney was markedly atrophic and fibrotic, consistent with unilateral end-stage kidney. The right ureter was markedly thickened, but with luminal continuity leading into the urinary bladder where a partial obstruction caused by nodular para-ureteral fat necrosis was evident. The lumen of the cutaneous fistula was continuous with the right ureter; therefore, we diagnosed the lesion as a ureterocutaneous fistula. Anomalies of the ureter are uncommon, and ureterocutaneous fistula formation in equids has not been reported previously to our knowledge.

Pharmacokinetics and Efficacy of the Benzothiazinone BTZ-043 against Tuberculous Mycobacteria inside Granulomas in the Guinea Pig Model.

Tuberculosis (TB), caused by Mycobacterium tuberculosis, is the world's leading cause of mortality from a single bacterial pathogen. With increasing frequency, emergence of drug-resistant mycobacteria leads to failures of standard TB treatment regimens. Therefore, new anti-TB drugs are urgently required. BTZ-043 belongs to a novel class of nitrobenzothiazinones, which inhibit mycobacterial cell wall formation by covalent binding of an essential cysteine in the catalytic pocket of decaprenylphosphoryl-ß-d-ribose oxidase (DprE1). Thus, the compound blocks the formation of decaprenylphosphoryl-ß-d-arabinose, a precursor for the synthesis of arabinans. An excellent in vitro efficacy against M. tuberculosis has been demonstrated. Guinea pigs are an important small-animal model to study anti-TB drugs, as they are naturally susceptible to M. tuberculosis and develop human-like granulomas after infection. In the current study, dose-finding experiments were conducted to establish the appropriate oral dose of BTZ-043 for the guinea pig. Subsequently, it could be shown that the active compound was present at high concentrations in Mycobacterium bovis BCG-induced granulomas. To evaluate its therapeutic effect, guinea pigs were subcutaneously infected with virulent M. tuberculosis and treated with BTZ-043 for 4 weeks. BTZ-043-treated guinea pigs had reduced and less necrotic granulomas than vehicle-treated controls. In comparison to the vehicle controls a highly significant reduction of the bacterial burden was observed after BTZ-043 treatment at the site of infection and in the draining lymph node and spleen. Together, these findings indicate that BTZ-043 holds great promise as a new antimycobacterial drug.

Dry-fog decontamination of microbiological safety cabinets after activities with SARS-CoV-2: cycle development and process validation for dry fogging with peroxyacetic acid.

Background: Technical protection measures for laboratory activities involving biological agents include biological safety cabinets (BSC) that may be contaminated. In the case of diagnostic activities with SARS-CoV-2, this may also affect BSC that are operated at protection level 2; therefore, decontamination of all contaminated surfaces of the BSC may be required. In addition to fumigation with hydrogen peroxide (H2O2), dry fogging of H2O2-stabilized peroxyacetic acid (PAA) represents another alternative to fumigation with formalin. However, to prove their efficacy, these alternatives need to be validated for each model of BSC. Methods: The validation study was performed on 4 different BSCs of Class II A2 using the "Mini Dry Fog" system. Results: An aerosol concentration of 0.03% PAA and 0.15% H2O2 during a 30 min exposure was sufficient to inactivate SARS-CoV-2. Effective concentrations of 1.0% PAA and 5% H2O2 were required to decontaminate the custom-prepared biological indicators loaded with spores of G. stearothermophilus and deployed at 9 different positions in the BSC. Commercial spore carriers were easier to inactivate by a factor of 4, which corresponded to a reduction of 106 in all localizations. Conclusions: Dry fogging with PAA is an inexpensive, robust, and highly effective decontamination method for BSCs for enveloped viruses such as SARS-CoV-2. The good material compatibility, lack of a requirement for neutralization, low pH - which increases the range of efficacy compared to H2O2 fumigation - the significantly shorter processing time, and the lower costs argue in favor of this method.

Technical and Anatomical Considerations for Reproducible Inactivation of Large Animal Carcasses by Steam Sterilization.

Introduction: The complete inactivation of infectious tissues of large animal carcasses is one of the most challenging tasks in high-containment facilities. Steam sterilization is a method frequently in use to achieve biological inactivation of liquid and solid waste. Objective: This study aims to highlight parameters most effective in creating reproducible cycles for steam sterilization of pig and calf carcasses. Methods: Two pigs or 1 calf were sterilized by running a liquid cycle (n = 3) at 121°C for at least 120 minutes in a pass-through autoclave. To assess the physical and biological parameters, temperature data loggers and biological indicators (BIs) with spores of Geobacillus stearothermophilus (ATCC 7953) were placed at defined positions within animal carcasses. After completion of each cycle, data loggers were analyzed and BIs were incubated for 7 days at 60°C. Results: Initial testing with an undissected pig carcass resulted in suboptimal temperatures at the tissue level with growth on 1 BI. After modifications of the used stainless-steel boxes and by placing the reference probe of the autoclave in the animal carcass, reproducible cycles could be created. A complete inactivation of BIs and a temperature profile of >121°C for at least 20 minutes could be achieved in almost all probed tissues. Conclusion: Only minor modifications in carcass preparation and the used sterilization equipment resulted in effective and reproducible cycles to inactivate large animal carcasses by using a steam autoclave.

Airborne Disinfection by Dry Fogging Efficiently Inactivates Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), Mycobacteria, and Bacterial Spores and Shows Limitations of Commercial Spore Carriers.

Airborne disinfection of high-containment facilities before maintenance or between animal studies is crucial. Commercial spore carriers (CSC) coated with 106 spores of Geobacillus stearothermophilus are often used to assess the efficacy of disinfection. We used quantitative carrier testing (QCT) procedures to compare the sensitivity of CSC with that of surrogates for nonenveloped and enveloped viruses, including severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), mycobacteria, and spores, to an aerosolized mixture of peroxyacetic acid and hydrogen peroxide (aPAA-HP). We then used the QCT methodology to determine relevant process parameters to develop and validate effective disinfection protocols (≥4-log10 reduction) in various large and complex facilities. Our results demonstrate that aPAA-HP is a highly efficient procedure for airborne room disinfection. Relevant process parameters such as temperature and relative humidity can be wirelessly monitored. Furthermore, we found striking differences in inactivation efficacies against some of the tested microorganisms. Overall, we conclude that dry fogging a mixture of aPAA-HP is highly effective against a broad range of microorganisms as well as material compatible with relevant concentrations. Furthermore, CSC are artificial bioindicators with lower resistance and thus should not be used for validating airborne disinfection when microorganisms other than viruses have to be inactivated.IMPORTANCE Airborne disinfection is not only of crucial importance for the safe operation of laboratories and animal rooms where infectious agents are handled but also can be used in public health emergencies such as the current severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic. We show that dry fogging an aerosolized mixture of peroxyacetic acid and hydrogen peroxide (aPAA-HP) is highly microbicidal, efficient, fast, robust, environmentally neutral, and a suitable airborne disinfection method. In addition, the low concentration of dispersed disinfectant, particularly for enveloped viral pathogens such as SARS-CoV-2, entails high material compatibility. For these reasons and due to the relative simplicity of the procedure, it is an ideal disinfection method for hospital wards, ambulances, public conveyances, and indoor community areas. Thus, we conclude that this method is an excellent choice for control of the current SARS-CoV-2 pandemic.

Absence of classical and atypical (H- and L-) BSE infectivity in the blood of bovines in the clinical end stage of disease as confirmed by intraspecies blood transfusion.

While the presence of bovine spongiform encephalopathy (BSE) infectivity in the blood of clinically affected sheep has been proven by intraspecies blood-transfusion experiments, this question has remained open in the case of BSE-affected cattle. Although the absence of infectivity can be anticipated from the restriction of the agent to neuronal tissues in this species, evidence for this was still lacking. This particularly concerns the production and use of medicinal products and other applications containing bovine blood or preparations thereof. We therefore performed a blood-transfusion experiment from cattle in the clinical end stage of disease after experimental challenge with either classical (C-BSE) or atypical (H- and l-) BSE into calves at 4-6 months of age. The animals were kept in a free-ranging group for 10 years. Starting from 24 months post-transfusion, a thorough clinical examination was performed every 6 weeks in order to detect early symptoms of a BSE infection. Throughout the experiment, the clinical picture of all animals gave no indication of a BSE infection. Upon necropsy, the brainstem samples were analysed by BSE rapid test as well as by the highly sensitive Protein Misfolding Cyclic Amplification (PMCA), all with negative results. These results add resilient data to confirm the absence of BSE infectivity in the donor blood collected from C-, H- and l-BSE-affected cattle even in the final clinical phase of the disease. This finding has important implications for the risk assessment of bovine blood and blood products in the production of medicinal products and other preparations.

Characterization of Experimental Oro-Nasal Inoculation of Seba's Short-Tailed Bats (Carollia perspicillata) with Bat Influenza A Virus H18N11.

In 2012 and 2013, the genomic sequences of two novel influenza A virus (IAV) subtypes, designated H17N10 and H18N11, were identified via next-generation sequencing in the feces of the little yellow-shouldered fruit bat (Sturnira lilium) and the flat-faced fruit-eating bat (Artibeus planirostris), respectively. The pathogenesis caused by these viruses in their respective host species is currently insufficiently understood, which is primarily due to the inability to obtain and keep these bat species under appropriate environmental and biosafety conditions. Seba's short-tailed bats (Carollia perspicillata), in contrast, are close relatives and a natural H18N11 reservoir species, with the advantage of established animal husbandry conditions in academic research. To study viral pathogenesis in more detail, we here oro-nasally inoculated Seba's short-tailed bats with the bat IAV H18N11 subtype. Following inoculation, bats appeared clinically healthy, but the histologic examination of tissues revealed a mild necrotizing rhinitis. Consistently, IAV-matrix protein and H18-RNA positive cells were seen in lesioned respiratory and olfactory nasal epithelia, as well as in intestinal tissues. A RT-qPCR analysis confirmed viral replication in the conchae and intestines as well as the presence of viral RNA in the excreted feces, without horizontal transmission to naïve contact animals. Moreover, all inoculated animals seroconverted with low titers of neutralizing antibodies.

Responsiveness of various reservoir species to oral rabies vaccination correlates with differences in vaccine uptake of mucosa associated lymphoid tissues.

Oral rabies vaccination (ORV) is highly effective in foxes and raccoon dogs, whereas for unknown reasons the efficacy of ORV in other reservoir species is less pronounced. To investigate possible variations in species-specific cell tropism and local replication of vaccine virus, different reservoir species including foxes, raccoon dogs, raccoons, mongooses, dogs and skunks were orally immunised with a highly attenuated, high-titred GFP-expressing rabies virus (RABV). Immunofluorescence and RT-qPCR screenings revealed clear differences among species suggesting host specific limitations to ORV. While for responsive species the palatine tonsils (tonsilla palatina) were identified as a main site of virus replication, less virus dissemination was observed in the tonsils of rather refractory species. While our comparison of vaccine virus tropism emphasizes the important role that the tonsilla palatina plays in eliciting an immune response to ORV, our data also indicate that other lymphoid tissues may have a more important role than originally anticipated. Overall, these data support a model in which the susceptibility to oral live RABV vaccine infection of lymphatic tissue is a major determinant in vaccination efficacy. The present results may help to direct future research for improving vaccine uptake and efficacy of oral rabies vaccines under field conditions.

Bat influenza viruses transmit among bats but are poorly adapted to non-bat species.

Major histocompatibility complex class II (MHC-II) molecules of multiple species function as cell-entry receptors for the haemagglutinin-like H18 protein of the bat H18N11 influenza A virus, enabling tropism of the viruses in a potentially broad range of vertebrates. However, the function of the neuraminidase-like N11 protein is unknown because it is dispensable for viral infection or the release of H18-pseudotyped viruses. Here, we show that infection of mammalian cells with wild-type H18N11 leads to the emergence of mutant viruses that lack the N11 ectodomain and acquired mutations in H18. An infectious clone of one such mutant virus, designated rP11, appeared to be genetically stable in mice and replicated to higher titres in mice and cell culture compared with wild-type H18N11. In ferrets, rP11 antigen and RNA were detected at low levels in various tissues, including the tonsils, whereas the wild-type virus was not. In Neotropical Jamaican fruit bats, wild-type H18N11 was found in intestinal Peyer's patches and was shed to high concentrations in rectal samples, resulting in viral transmission to naive contact bats. Notably, rP11 also replicated efficiently in bats; however, only restored full-length N11 viruses were transmissible. Our findings suggest that wild-type H18N11 replicates poorly in mice and ferrets and that N11 is a determinant for viral transmission in bats.

Genetic Characterization and Zoonotic Potential of Highly Pathogenic Avian Influenza Virus A(H5N6/H5N5), Germany, 2017-2018.

We genetically characterized highly pathogenic avian influenza virus A(H5N6) clade 2.3.4.4b isolates found in Germany in 2017-2018 and assessed pathogenicity of representative H5N5 and H5N6 viruses in ferrets. These viruses had low pathogenicity; however, continued characterization of related isolates is warranted because of their high potential for reassortment.

Characterization of protective humoral and cellular immune responses against RHDV2 induced by a new vaccine based on recombinant baculovirus.

Rabbit hemorrhagic disease (RHD) is a lethal disease in rabbits caused by RHD virus (RHDV). Protection is only possible through vaccination. A new virus variant (RHDV2) which emerged in 2010 in France differed from the classical RHDV1 variant in certain aspects and vaccines against RHDV1 induced limited cross protection only. In a previous study, we designed a recombinant baculovirus based RHDV2-VP1 vaccine, which provided a protective immunity in rabbits against RHDV2. In the present study this newly created vaccine is characterized with regard to onset and duration of protection, and possible cross protection against classical RHDV1. Furthermore, humoral and cellular immune mechanisms in vaccinated and infected rabbits were analyzed. In all experiments, the recombinant vaccine was compared to a conventional liver-based RHDV2 vaccine. The RHDV2-VP1 vaccine induced a protective immune response already seven days after single vaccination and fully protected for at least 14 months. A booster vaccination 21 days after the first had a negative influence on long-term protection. The cross protection provided by the RHDV2-VP1 vaccine against classical RHDV1 was limited since only 50% of vaccinated rabbits survived the infection. Conclusively, the new, baculovirus-based RHDV2-VP1 vaccine has the potential to protect rabbits against the infection with RHDV2, blocks completely the disease progression and prevents the spread of RHDV2 at the population level.

Double-attenuated influenza virus elicits broad protection against challenge viruses with different serotypes in swine.

Influenza A viruses (IAV) have caused seasonal epidemics and severe pandemics in humans. Novel pandemic strains as in 2009 may emerge from pigs, serving as perpetual virus reservoir. However, reliably effective vaccination has remained a key issue for humans and swine. Here, we generated a novel double-attenuated influenza live vaccine by reverse genetics and subjected immunized mice and pigs to infection with the homologous wild-type, another homosubtypic H1N1, or a heterosubtypic H3N2 virus to address realistic challenge constellations. This attenuated mutant contains an artificial, strictly elastase-dependent hemagglutinin cleavage site and a C-terminally truncated NS1 protein from the IAV A/Bayern/74/2009 (H1N1pdm09). Prior to challenge, we immunized mice once and pigs twice intranasally. In vitro, the double-attenuated mutant replicated strictly elastase-dependently. Immunized mice and pigs developed neither clinical symptoms nor detectable virus replication after homologous challenge. In pigs, we observed considerably reduced clinical signs and no nasal virus shedding after homosubtypic and reduced viral loads in respiratory tracts after heterosubtypic infection. Protection against homosubtypic challenge suggests that an optimized backbone strain may require less frequent updates with recent HA and NA genes and still induce robust protection in relevant IAV hosts against drifted viruses.

MHC class II proteins mediate cross-species entry of bat influenza viruses.

Zoonotic influenza A viruses of avian origin can cause severe disease in individuals, or even global pandemics, and thus pose a threat to human populations. Waterfowl and shorebirds are believed to be the reservoir for all influenza A viruses, but this has recently been challenged by the identification of novel influenza A viruses in bats1,2. The major bat influenza A virus envelope glycoprotein, haemagglutinin, does not bind the canonical influenza A virus receptor, sialic acid or any other glycan1,3,4, despite its high sequence and structural homology with conventional haemagglutinins. This functionally uncharacterized plasticity of the bat influenza A virus haemagglutinin means the tropism and zoonotic potential of these viruses has not been fully determined. Here we show, using transcriptomic profiling of susceptible versus non-susceptible cells in combination with genome-wide CRISPR-Cas9 screening, that the major histocompatibility complex class II (MHC-II) human leukocyte antigen DR isotype (HLA-DR) is an essential entry determinant for bat influenza A viruses. Genetic ablation of the HLA-DR α-chain rendered cells resistant to infection by bat influenza A virus, whereas ectopic expression of the HLA-DR complex in non-susceptible cells conferred susceptibility. Expression of MHC-II from different bat species, pigs, mice or chickens also conferred susceptibility to infection. Notably, the infection of mice with bat influenza A virus resulted in robust virus replication in the upper respiratory tract, whereas mice deficient for MHC-II were resistant. Collectively, our data identify MHC-II as a crucial entry mediator for bat influenza A viruses in multiple species, which permits a broad vertebrate tropism.

White-Tailed Sea Eagle (Haliaeetus albicilla) Die-Off Due to Infection with Highly Pathogenic Avian Influenza Virus, Subtype H5N8, in Germany.

In contrast to previous incursions of highly pathogenic avian influenza (HPAIV) H5 viruses, H5N8 clade 2.3.4.4b viruses caused numerous cases of lethal infections in white-tailed sea eagles (Haliaeetus albicilla) affecting mainly young eagles (younger than five years of age) in Germany during winter 2016/2017. Until April 2017, 17 HPAIV H5N8-positive white-tailed sea eagles had been detected (three found alive and 14 carcasses) by real-time RT-PCR and partial nucleotide sequence analyses. Severe neurological clinical signs were noticed which were corroborated by immunohistopathology revealing mild to moderate, oligo- to multifocal necrotizing virus-induced polioencephalitis. Lethal lead (Pb) concentrations, a main factor of mortality in sea eagles in previous years, could be ruled out by atomic absorption spectrometry. HPAIV H5 clade 2.3.4.4b reportedly is the first highly pathogenic influenza virus known to induce fatal disease in European white-tailed see eagles. This virus strain may become a new health threat to a highly protected species across its distribution range in Eurasia. Positive cloacal swabs suggest that eagles can spread the virus with their faeces.

A novel European H5N8 influenza A virus has increased virulence in ducks but low zoonotic potential.

We investigated in a unique setup of animal models and a human lung explant culture biological properties, including zoonotic potential, of a representative 2016 highly pathogenic avian influenza virus (HPAIV) H5N8, clade 2.3.4.4 group B (H5N8B), that spread rapidly in a huge and ongoing outbreak series in Europe and caused high mortality in waterfowl and domestic birds. HPAIV H5N8B showed increased virulence with rapid onset of severe disease and mortality in Pekin ducks due to pronounced neuro- and hepatotropism. Cross-species infection was evaluated in mice, ferrets, and in a human lung explant culture model. While the H5N8B isolate was highly virulent for Balb/c mice, virulence and transmissibility were grossly reduced in ferrets, which was mirrored by marginal replication in human lung cultures infected ex vivo. Our data indicate that the 2016 HPAIV H5N8B is avian-adapted with augmented virulence for waterfowl, but has low zoonotic potential. The here tested combination of animal studies with the inoculation of human explants provides a promising future workflow to evaluate zoonotic potential, mammalian replication competence and avian virulence of HPAIV.

Outbreaks among Wild Birds and Domestic Poultry Caused by Reassorted Influenza A(H5N8) Clade 2.3.4.4 Viruses, Germany, 2016.

In November 2016, an influenza A(H5N8) outbreak caused deaths of wild birds and domestic poultry in Germany. Clade 2.3.4.4 virus was closely related to viruses detected at the Russia-Mongolia border in 2016 but had new polymerase acidic and nucleoprotein segments. These new strains may be more efficiently transmitted to and shed by birds.